Extended Data Fig. 9: MLH1 knock-out cell line validation and EGFR-targeting epegRNA library design.
From: Multimodal scanning of genetic variants with base and prime editing
(a) Venn diagram showing the numbers of EGFR coding variants listed in the ClinVar and COSMIC databases that can be introduced by the ABE8e and BE3.9max base editors. All possible sgRNAs with an NGG PAM targeting EGFR exons were considered and editing outcomes were predicted using the base editor design tool from Hanna et al.33. (b,c) (b) Western Blot analysis of MLH1 protein expression and (c) Sanger sequencing of the MLH1 endogenous gene locus in two clonal cell lines derived from the transfection of MCF10A cells with a plasmid expressing the Cas9 enzyme and a sgRNA targeting MLH1 exon 2. Clone 3 was used for all subsequent experiments. The data are representative of a single experiment. (d) Bar plot representing the distance between the nCas9-induced nick and intended edit across the pegRNA library. A distance of zero corresponds to deletions overlapping the nick-adjacent nucleotide. (e) Histogram representing the number of pegRNAs attributed to each unique edit in the library. The pegRNAs sharing the same intended mutations with or without additional synonymous mutation are considered separately. (f) Bar plot representing the number of pegRNAs in the library according to the type of introduced mutation. Intended edits are, when possible, associated with an additional synonymous mutation (intended + synonymous). For each of these, control pegRNAs introducing only the intended edit (intended edit only) or the synonymous mutation (synonymous only) are also added to the library. Some edits can correspond to both intended and synonymous mutations (both intended and synonymous). (g) Library distributions and sample correlations for the prime editing EGFR activation screen in MCF10A cells. Raw barcode counts and Person correlation coefficients are shown for all samples.
